Highly pathogenic avian influenza (HPAI) H5N1 viruses and their capacity for transmission from birds to humans have raised worldwide concerns about a potential forthcoming human pandemic. With the continued spread of H5N1 influenza virus, new virus strains have emerged and will continue to change and evolve in the future. The World Health Organization has classified the H5N1 viruses isolated recently into 10 clades (or sublineages) based on the phylogenetic analysis of viral hemagglutinin (HA) sequences of H5N1 viruses. With the continuous threat of a new influenza pandemic arising from avian reservoirs, the development of broadly protective vaccines is particularly important. To date, the broadly protective H5N1 vaccines have been mainly achieved using novel adjuvant formulations.
However, the inherent nature of influenza virus antigenic changes has not been taken into accounts in the immunogen designs for developing broadly protective H5N1 vaccines. Refocusing antibody responses have been proposed by designing the immunogens that can preserve the overall fold of the immunogen structure but selectively mutate the “undesired” antigenic sites that are highly variable (escape mutants evade protective immune responses), immunosuppressive (downregulate the immune response to the infection), cross-reactive (the immune response induces a reaction to a protein resembling the immunogen). The immunogen design by refocusing antibody responses has been applied for HIV-1 vaccines using the hyperglycosylated HIV-1 gp120 immunogens where the undesired eptiopes are masked by selective incorporations of N-linked glycans. The glycan masking strategy has been also recently reported to design influenza virus vaccines that can enhance the antibody responses against a broad range of H3N2 intertypic viruses. However, there is no report for the use of glycan-masking immunogen design for H5N1 vaccines.
DNA vaccine has been considered as the revolutionary vaccinology with the advantages in offering genetically antigen design, time to manufacturing, long stability without the need for cold chains supply, and the immunogenicity predominantly elicited by T cells through the endogenerous antigen processing pathways. However, the apparent low immunogenicity of DNA vaccines in large animals (including humans) has been overcome using novel delivery systems such as gene-guns or electroporation. Additionally, the DNA vaccine-elicited immune responses can be further augmented using the heterologous prime-boost immunization regimen where the booster dose uses a different vaccine format containing the same or similar antigens. Examples of DNA vaccine prime-boost immunization strategy has been reported for the inactivated influenza virus, live-attenuated influenza virus, recombinant adenovirus, virus-like particles (VLPs) and recombinant subunit proteins in adjuvants. Furthermore, human vaccines receiving the H5 DNA vaccine priming followed by a booster with inactivated H5N1 vaccine were found to enhance the protective antibody responses (HAI) and in some cases induce the haemagglutinin-stem-specific neutralizing antibodies.
Influenza VLPs are noninfectious and have a size and morphology that are similar to those of native virion structures, but they do not contain the genomic RNAs for virus replication. The assembly of influenza VLPs depends on the interactions of M1 proteins and/or other viral surface proteins, such as HA, NA, and M2, with the cellular lipid membranes. The interactions of M1 protein with the cytoplasmic tails of HA and NA spikes can increase the lipid membrane binding of M1 proteins in assembling influenza virus. The interactions of HA and NA with the M1 protein can also reduce the formation of elongated intracellular immature particles and improve the secretion of spherical mature VLPs. Additionally, the cytoplasmic tails of M2 protein, by interacting with the M1 protein, further promote the budding and release of the influenza virions. Recently, the M2 protein was found to act as the plasma membrane-targeting signal for the budding and egress of influenza virions. Host cell proteins can be recruited into the VLPs, as recently shown by LC/MS/MS analyses. Therefore, the biosynthesis of influenza VLPs is a self-assembly process that involves complex interactions of viral and cellular components.
There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades.